Hepatitis b virus pre-s1 derived synthetic polypeptides and uses thereof

ABSTRACT

The invention relates to a group of synthetic polypeptides, derived from the pre-S1 region of HBV, that efficiently interfere with early steps of an HBV infection. The peptides of the invention can be used in diagnostics for the detection of antigens and/or antibodies.

This application is a continuation application of U.S. application Ser.No. 13/014,070, filed Jan. 26, 2011, which is a divisional applicationof U.S. application Ser. No. 11/641,066, filed Dec. 19, 2066, which is acontinuation application of U.S. application Ser. No. 10.484,923, havinga U.S. filing date of Jul. 13, 2004, the date of completion of allrequirements under 35 U.S.C. 371, and which is a National Stageapplication of PCT/IB02/02915, filed Jul. 26, 2002, all of saidapplications incorporated herein by reference.

The present invention relates to new polypeptides and derivativesthereof that block Hepatitis B infection at an early step.

Hepadnaviruses are a group of small-enveloped partially double strandedDNA viruses, which have been found in mammals and birds. The humanhepatitis B virus (HBV), representing the prototype of this group,causes acute and chronic hepatitis in man. Infections with the HBVrepresent a severe health problem, leading to an estimated one milliondeath per year.

The intracellular replication cycle of hepadnaviruses, in particulartranscription of the viral DNA, packaging and reverse transcription ofgenomic RNA and establishment of an intracellular pool of covalentlyclosed circular HBV-DNA has been elucidated in some detail (Nassal etal, 1993; Nassal et al, 1996). However, until now the lack of anHBV-infectable cell line and the restricted availability of primaryhuman hepatocytes for in vitro infection studies relentlessly preventedprogress in the understanding of the early steps of the HBV-infection.Accordingly a variety of HBV-receptor candidates have been proposed onthe bases of binding studies (reviewed by DeMeyer et al, 1997), but noneof them could be convincingly demonstrated to be involved in the entryprocess.

However, more progress has been made recently in determining thesequence requirements for infection in the envelope proteins of duck andhuman hepatitis B virus (Urban et al., 1998, LeSeyec et al., 1998,LeSeyec et al., 1999).

The virus shell of HBV contains the large (L-), the middle (M-) and thesmall (S-) viral envelope proteins. Avian hepadnaviruses comprise onlyL- and S-protein. The surface proteins of all hepadnaviruses are encodedin single open reading frames of the viral DNA such that the L-proteincontains the complete S-domain which serves as an anchor in the ERmembrane-derived lipid bilayer. In the case of HBV, L- and M-proteinsdiffer from S-protein in an hydrophilic N-terminal extension of 163 to174 amino acids, termed pre-S. This pre-S extension is further dividedinto a pre-S2 (55 amino acids) and a pre-S1 (108 amino acids in subtypeayw) domain, towards the amino terminus. Depending on the HBV subtype,the pre-S1 domain shows some sequence variation and in case of subtypeadr carries an insertion of additional 11 amino acids.

The analysis of the infectivity of pseudotyped HBVs carrying mutationsin the exterior pre-S part of the L-protein has shown that the infectiondepends on an extended sequence within the pre-S1 domain of the HBVL-protein (Le Seyec, 1999). In contrast, major parts of the pre-S2domain turned out to be dispensable for infectivity (Le Seyec et al,1998). This indicates that the sequence between amino acids 3 and 77 ofthe pre-S1 domain is involved in the infection step.

Due to the availability of primary duck hepatocytes more progress hasbeen achieved in the related duck hepatitis B virus model system. Usingthis system it was shown that E. coli-derived preS-polypeptides of theduck hepatitis B virus L-protein inhibit DHBV-infection of primary duckhepatocytes by interfering with receptor binding (Urban et al., 1998).It was further demonstrated that the DHBV-preS binding proteinCarboxypeptidase D, initially identified by Kuroki et al., (1994)functions as a common receptor component for avian HBVs (Urban et al,1998; Breiner et al, 1998; Urban et al, 2000).

In the case of HBV infection, a similar state of knowledge is stillmissing and there is yet no experimental evidence for peptide componentsthat efficiently interfere with early steps of an HBV-infection.

The authors of the present invention have investigated whetherrecombinant, E. coli derived preS-polypeptides and chemicallysynthesized preS-peptides of HBV L-protein could interfere with aHBV-infection.

They have identified a group of synthetic peptides that exhibits at thesame time an excellent direct inhibitory activity against HBV infectionand makes it possible to elicit immune protection against HBV.

These polypeptides were designed from the sequence of the pre-S1 aminoacids 1 to 78 (SEQ ID NO: 1) of the Hepatitis B virus subtype ayw.However, although these polypeptides are shorter than the pre-S1 region,they surprisingly show a better inhibitory activity than longerpolypeptides that would mimic the entire pre-S1 region.

The isolated synthetic polypeptides, or variant thereof, of the presentinvention have an amino acid sequence of the formula:

X-Y-Z  (I)

wherein:

-   -   X is an amino acid, for instance a methionine, or absent;    -   Y is the amino acid sequence 2 to 48 of pre-S1 region shown in        SEQ ID NO: 2, or a N-terminally and/or C-terminally truncated        form of this sequence, or variants thereof;    -   Z, linked to the —CO— group of the last residue of Y, is an        amino acid sequence comprising 1 to 30 consecutive amino acids        from the pre-S1 region shown in SEQ ID NO: 3, or absent; said        polypeptides being optionally chemically modified to bear a        hydrophobic moiety.

Preferably, when Y is a C-terminally, and optionally N-terminally,truncated form of sequence SEQ ID NO: 2, or a variant thereof, Z isabsent.

Preferably, the hydrophobic moiety corresponds to the acyl rest of asaturated or unsaturated fatty acid having at least 4 carbon atoms,preferably at least 6 carbon atoms, more preferably at least 8 carbonatoms, such as myristic acid, palmitic acid, stearic acid, oleate,linoleate, linolenate or arachidonate for instance, or the hydrophobicmoiety corresponds to a cholesterol group or the like.

In a preferred embodiment, the polypeptides of the present invention aremyristoylated. More preferably, the polypeptides comprise the entireamino acid sequence from 2 to 48 of SEQ ID NO: 1, amino acid 2 (glycine)carrying a myristyl group:Myr-GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVG (Myr 2-48, SEQ IDNO: 7).

In another embodiment, the polypeptide of the invention has the sequenceSEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 7.

The term “variant” refers to the homologous polynucleotidic sequencesfound in the different viral species, strains or subtypes of thehepadnavirus genus, such as HBV strain alpha1, HBV strain LSH(chimpanzee isolate), woodchuck HBV, Woolly Monkey HBV (WMHBV), orstrains selected from the group consisting of the HBV subtypes AD, ADR,ADW, ADYW, AR and AYW.

Any analogs to the pre-S1 synthetic polypeptides are part of the presentinvention. Analogs involve amino acid deletions, amino acidsubstitutions such as conservative or non conservative replacement byother amino acids or by isosteres (modified amino acids that bear closestructural and spatial similarity to protein amino acids), amino acidadditions or isostere additions, as long as the sequences elicit 70%inhibition of human hepatocyte primary cultures HBV infection with apeptide concentration below 100 μM, preferably below 10 μM, andpreferably below 1 μM.

Conservative amino acid substitutions typically relates to substitutionsamong amino acids of the same class. These classes include, for example,amino acids having uncharged polar side chains, such as asparagine,glutamine, serine, threonine and tyrosine; amino acids having basic sidechains, such as lysine, arginine, and histidine; amino acids havingacidic side chains, such as aspartic acid and glutamic acid; and aminoacids having nonpolar side chains, such as glycine, alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, andcysteine.

Advantageously, peptides according to the invention are modified toresist proteolysis. This can be achieved for instance by substitutingL-amino acids at exposed sites with their D-amino acid counterpart.

In another embodiment, the polypeptides of the invention can incorporateshort peptidic or non peptidic linkers so as to designthree-dimensionally constrained peptides (Root M. J. et al., 2001).

The polypeptides of this invention can be prepared by a variety ofprocedures readily known to those skilled in the art. Such proceduresinclude more particularly the solid phase sequential and block synthesis(B. W. Erickson and R. B. Merrifield, 1976).

The solid phase sequential procedure can be performed using establishedautomated methods such as by use of an automated polypeptidesynthesizer. In this procedure an α-amino protected amino acid is boundto a resin support. The resin support employed can be any suitable resinconventionally employed in the art for the solid phase preparation ofpolypeptides, preferably polystyrene which has been copolymerized withpolyoxyethylen to provide sites for ester formation with the initiallyintroduced o-amino protected amino acid. This optimized method, appliedby the inventors, has been explicitly described by Gausepohl et al, 1989and 1990.

The amino acids are introduced one by one. Each synthesis cyclecorresponding to the introduction of one amino acid includes adeprotection step, successive washing steps, a coupling step withactivation of the amino acid, and subsequent washing steps. Each ofthese steps is followed by a filtration.

The reactive agents for coupling are the classical reactive agents forpolypeptide synthesis such as dicyclohexylcarbodiimide,hydroxybenzotriazole, benzotriazil-1-yl-oxytris (dimethylamino)phosphonium hexafluorophosphate, and diphenylphosphorylazide.

After synthesis of the polypeptide on the resin, the polypeptide isseparated from the resin by a treatment with a strong acid such astrifluoroacetic acid in the presence of anisole, ethanedithiol or2-methylindole. The compound is then purified by the classicaltechniques of purification, in particular by means of high pressureliquid chromatography (HPLC).

The polypeptides of the present invention may also be obtained bycoupling polypeptide fragments that are selectively protected, thiscoupling being effected in a solution.

The polypeptide can further be produced by genetic engineeringtechniques. An eukaryotic expression system, such as the baculovirussystem, is particularly suitable. According to this procedure proteinsare expressed in insect cells infected with a recombinant baculoviruscontaining a nucleic acid sequence encoding a heterologous protein andregulating nucleic acid sequences, such as a promoter. Severalcell-lines are available for infection with recombinant baculovirus,such as cell line SF-9, available from the American Type CultureCollection (CRL 1711).

The polypeptides of the present invention can be used to inhibit invitro or in vivo hepatocyte infection by HBV through preventing bindingand/or internalisation of HBV particles to hepatocytes.

Accordingly, the invention provides a method of in vitro inhibition ofhepatocyte infection by HBV comprising using a polypeptide as describedabove.

Suitable hepatocytes include human primary hepatocytes or the hepatomaderived cell line called HepaRG (described in the patent application FR0109044), which is also susceptible to HBV infection.

The invention further relates to a method for in vitro and/or in vivoidentification of a hepatocyte receptor involved in the attachmentand/or penetration of HBV and/or quantitation of the expression of saidreceptor that comprises using a peptide as described above.

In particular, said method comprises the steps consisting of:

-   -   contacting a liver biopsy or a hepatocyte with a polypeptide of        the invention under conditions and for a period of time        sufficient to allow specific binding of said polypeptide to a        receptor expressed at the surface of a hepatocyte;    -   detecting binding of said polypeptide to a receptor; and    -   identifying said receptor.

This can be achieved according to classical procedures well-known by theskilled in the art. For instance, this could involve radioactive, enzymeor fluorescent labelling of the polypeptides of the invention, andsubsequent detection with an appropriate method. A number of fluorescentmaterials are known and can be utilized as labels. These include, forexample, fluorescein, rhodamine, auramine, Texas Red. Enzyme labelsconsists in conjugation of an enzyme to a molecule of interest, e.g. apolypeptide, and can be detected by any of colorimetric,spectrophotometric, or fluorospectrophotometric techniques.

The present invention also relates to antibodies directed against apolypeptide according to the invention, or a fragment of saidpolypeptide. The antibodies of the present invention can be chimericantibodies, humanized antibodies, or antigen binding fragments Fab,Fab′, F(ab′)₂ and Fv. They can also be immunoconjugated or labelledantibodies.

Polyclonal antibodies can be obtained from serum of an animal immunizedagainst a polypeptide of the invention, according to standard methodswell-known by one skilled in the art. The specificity of the polyclonalserum can be further increased through immuno-affinity chromatographyusing polypeptides of the formula (I), or fragment thereof, immobilisedon a solid phase. The antibody is contacted with the immobilisedimmunising polypeptide during a time sufficient to allow the formationof a complex antibody-immobilised polypeptide.

Monoclonal antibodies can be obtained according to the standard methodof hybridoma culture (Kohler and Milstein, 1975).

Said antibodies are particularly useful for inhibiting in vitro or invivo HBV infection of hepatocytes, or for ex vivo assessing theinfectivity of HBV particles.

Accordingly, the invention provides a method for assessing ex vivo theinfectivity of HBV particles comprising using an antibody according tothe invention.

In another embodiment, the polypeptides or antibodies of the inventioncan be used as a drug. Such a drug can be particularly useful forblocking or preventing HBV infection.

Pharmaceutical compositions comprising, in a pharmaceutical acceptablecarrier, either a polypeptide or an antibody according to the inventionare within the scope of the invention.

Accordingly, the invention provides a therapeutical method for theprevention or the treatment of HBV infection that comprisesadministration of a peptide or antibody of the invention to a patient inneed thereof.

The invention further relates to the use of a polypeptide or an antibodyof the invention for the manufacture of a medicament intended for the invivo inhibition of HBV infection. As used herein, the term “treatment”is intended for the improvement or the stabilisation of the condition ofa patient. This include for instance prevention of HBV propagation touninfected cells of an organism.

The invention further provides a therapeutical composition thatcomprises a polypeptide or antibody of the invention associated to apharmaceutically acceptable carrier.

In another aspect, the invention relates to a method of vaccinationagainst HBV infection that comprises administration of a peptide of theinvention to a patient in need thereof.

Accordingly, the present invention includes a vaccinal compositioncomprising, in a pharmaceutically acceptable carrier, a polypeptidedisclosed herein.

In the context of the present application, “vaccination” is intended forprophylactic or therapeutical vaccination. “Therapeutical vaccination”is meant for vaccination of a patient with HBV infection.

In further aspect, the invention provides a immunogenic compositioncomprising in a pharmaceutical acceptable support, a polypeptidedisclosed herein.

Preferably, the peptide(s) of the vaccine method/composition orimmunogenic composition is myristoylated. Myristoylation indeed makes itpossible to enhance the immune response elicited toward the peptideantigen.

The polypeptides of the invention can be modified to have increasedimmunogenic properties. Such increased immunogenic properties refer forinstance to increasing the range of antibodies elicited followingimmunization, or to allowing the production of antibodies capable ofneutralizing infection by various viral strains.

In another embodiment, the polypeptides of the invention can be modifiedto decrease their immunogenic properties. Such polypeptides would beparticularly useful in a therapeutical application to inhibit in vivoHBV infection while avoiding or limiting adverse effects.

A “pharmaceutically acceptable carrier” refers to any vehicle whereinthe pharmaceutical or vaccine compositions according to the inventionmay be formulated. It includes a saline solution such as phosphatebuffer saline. In general, a diluent or carrier is selected on the basisof the mode and route of administration, and standard pharmaceuticalpractice.

According to the invention, the term “patient” is meant for any humanlikely to be infected with HBV.

Polypeptides of the invention can be used for ex vivo diagnosis of HBVinfection through detecting the interaction of said polypeptides withantibodies present in a biological sample and directed against afragment of the pre-S1 region of HBV viral particles. The specificinteraction of said polypeptides with endogenous antibodies can bedetected by any suitable detection method readily known by the skilledin the art.

The invention thus provides a method for ex vivo diagnosis of HBVinfection comprising the steps consisting of:

-   -   contacting a biological sample with a polypeptide of the        invention under conditions and for a period of time sufficient        to allow formation of complexes between said polypeptide and an        antibody present in a biological sample and directed against a        fragment of the pre-S1 region of HBV viral particles;    -   detecting said complexes, the presence of which is indicative of        HBV infection.

The diagnosis method of the invention is useful as a predictive methodof HBV infection development. Accordingly, repetitions at differentpoints in time of the above ex vivo diagnosis method make it possible toreveal an increase or a decrease in the number of antibodies against afragment of the pre-S1 region of HBV viral particles that are detected.An increase in the number of said antibodies usually indicates animprovement of the patient's condition. In particular, such a methodallows assessment of a patient's response to a treatment.

Detection can be achieved through any means, e.g. radioactive, enzyme orfluorescent labelling of the polypeptides of the invention combined withappropriate means of detection, readily known by one skilled in the art.

The present invention is also directed to diagnostic methods relating tothe detection antibodies to pre-S1 gene-encoded HBV antigens. Suchantibodies can be detected ex vivo in a biological sample with thesynthetic peptidic immunogens disclosed herein by both sandwich typeimmunoassays and competition type immunoassays, such as thoseimmunoassays in which antigen in the sample competes with labelledimmunogen for antibody.

Therefore, the invention further concerns a method for detecting a HBVinfection comprising effecting quantitative immunoassays on a serumtaken form a human to determine the amount of antibodies presenttherein, which are antibodies to an antigen coded by the pre-S1 region,employing the above-described HBV peptide immunogen and comparing thevalue with a known standard.

The invention also relates to a method for detecting the presence of HBVinfection comprising effecting quantitative immunoassays on a serumtaken form a human to determine the amount of antigens coded by thepre-S1 region, employing the above-described antibodies to the HBVpeptide immunogen and comparing the value with a known standard.

Both peptides or antibodies according to the invention can similarly beused to determine the outcome of HBV infection by periodically carryingout an immunoassay on a biological sample from a patient so as to assessthe amount of antibodies to an antigen coded by the pre-S1 region, orantigens coded by the pre-S1 region.

The invention will be further understood in view of the followingexamples and the annexed figures wherein:

FIG. 1 is the comparison of HBV infectivity of human hepatocytescultured in the presence of a myristoylated or non myristoylated peptidecorresponding to a pre-S1 region domain (1-78). The infection efficiencywas evaluated by measuring HBsAg in the supernatant of infected cells,14 days post-infection. Values are expressed as a % of the control (nopeptide).

FIG. 2 illustrates the comparison of inhibitory activity of C-terminallytruncated myristoylated peptides. Human hepatocytes were HBV infected inthe presence of myristoylated or non-myristoylated peptidescorresponding to parts of the Pre-S1 domain of the HBV L protein. Theconcentration of peptides ranged from 0.8 to 800 nM. Infectionefficiency was evaluated by measuring HBsAg secretion 7 days afterinfection and expressed as a % of the positive control.

FIG. 3 is the comparison of the inhibitory activity of C-terminallytruncated peptides shorter than Myr 2-48. The concentration of peptidesranged from 0.8 to 8 μM. Infection efficiency was evaluated by measuringHBsAg secretion 8 days after infection and expressed in ng/ml.

FIG. 4 illustrates the comparative inhibitory activity of WMHBV and HBVderived myristoylated 2-48 peptides. Infection of human primary humanhepatocytes with HBV was performed in the presence of decreasingconcentration of peptides. The supernatant of infected cells wascollected at day 10 post-infection and analysed for the presence ofHbsAg.

FIG. 5 is a Dot Blot analysis of duck HBV (DHBV) in the serum of duckstreated with duck preS Myr 2-41 (DpreS2-41^(myr)), heron preS Myr 2-44(HepreS2-44^(myr)), human preS Myr 2-68 (HupreS2-68^(myr)), duck preSMyr 2-21 (DpreS2-21^(myr)) or ddH₂O, 5, 9, 15 and 28 days post-infection(p.i.) with DHBV.

FIG. 6 is a Western Blot analysis of DHBV L protein in the serum ofducks 35 days post infection.

EXAMPLE 1 Material and Methods for the Inhibition of HBV Infection withpre-S1-HBV Synthetic Polypeptides

a) Establishment of HBV-Infectable Cell Culture

Fragments of normal adult human liver were obtained from patientsundergoing hepatic resection for liver metastases (the fragments weretaken at a distance from the metastasis in macroscopically normalliver). Access to this biopsy material was in agreement with French lawsand satisfied the requirements of the French National Ethics Committee.Hepatocytes were isolated by the procedure of Guguen-Guillouzo andGuillouzo and cultured in H medium supplemented with 3.5×10⁻⁶ Mhydrocortisone hemisuccinate, 2 mM L-glutamine, 50 mg of gentamicin perlitre, 2% dimethyl sulfoxide, 5% adult human serum, and 5% FCS. Threedays after seeding, the cells were infected.

Alternatively, for some experiments, instead of using primary cultures,we made use of a new hepatoma derived cell line, called HepaRG, which isalso susceptible to HBV infection (patent application FR 0109044).Before the infection procedure cells were allowed to differentiate,allowing cells to gain an hepatocyte-like morphology (patent applicationFR 0109044). Cells were then infected.

b) HBV Infection of Cell Culture

As an infectious inoculum, a 50-fold concentrated culture medium ofHepG2 clone 2.2.15 cells was used, because of an unlimited supply and aconstant quality.

It was prepared as described previously. Differentiated cells wereincubated with the concentrated infectious source, 10-fold diluted inculture medium supplemented with 5% PEG 8000 (Sigma), for 20 h at 37° C.as described previously (Gripon et al., 1988; Gripon et al., 1993).Control cultures were incubated with 5% PEG and 25% FCS diluted inphosphate-buffered saline (PBS) instead of the infectious source. At theend of the incubation, cells were washed three times with the culturemedium and maintained in the presence of 2% DMSO and 5×10⁻⁵ Mhydrocortisone hemisuccinate and harvested at indicated times.

c) Polypeptide Competition Assays

Polypeptide competition assays were performed by pre-incubating cellswith the analyzed polypeptide for 30 min, at 37° C., prior to theaddition of the infectious source.

d) Inhibition of Hbv Infection Assessment

HBsAg Assay

HBsAg was detected in the medium by an ELISA kit (Monolisa AgHBs plus)obtained from Bio-Rad Laboratories. Values are expressed in ng/ml ofsupernatant or percent of control (absence of peptide).

RNA Extraction and Analysis

Total cellular RNA was extracted by Total SV RNA kit (Promega, France),fractionated on a 1.5% agarose gel and analyzed by standard Northernblot procedure (Sambrook et al., 1989). Control of the RNA amounttransferred onto filters was performed after methylene blue staining.Hybridization was performed with P³² labeled HBV DNA.

EXAMPLE 2 Results of HBV Infection Inhibition with Pre-S1-HBV SyntheticPolypeptides

a) Influence of the Myristoylation of Pre-S1 Synthetic Peptides on HBVInfection Inhibition

Mutagenesis experiments have previously shown that a part (amino acids(AA) 3-77) of the pre-S1 region was essential for HBV infectivity (LeSeyec et al., 1999). In addition we have also demonstrated thatmyristoylation of the AA 2, a glycine residue, associated with theremoval of AA 1, a methionine residue, was also critical (Gripon et al.,1995). We have therefore postulated that a peptide comprising aminoacids 2-77, with a myristoylated glycin, could interfere with the HBVinfection process. To evaluate this hypothesis two peptides weresynthesized: PreS1-78 and Myr PreS 2-78. These peptides were then addedprior and during the infection process of human hepatocyte cultures, theinfection level was evaluated by measuring the HBsAg secretion ofinfected cells. FIG. 1 displays that although the non-myristoylatedpeptide has only a faint effect on HBV infectivity at 1 μM, the sameamount of the myristoylated peptide did almost completely abolish it,lower doses were partly inhibitory. These results were confirmed by RNAanalysis.

Other experiments conducted with higher peptide concentrations (up to100 μM) indicate that myristoylation is not absolutely required for theinhibition of HBV infection but strongly enhances the activity of thepeptides by a factor of about 100 fold.

b) Activity of C-Terminally Truncated Pre-S1 Synthetic Peptides

Human hepatocytes were HBV infected in the presence of myristoylated ornon-myristoylated peptides corresponding to parts of the Pre-S1 domainof the HBV L protein. The concentration of peptides ranged from 0.8 to800 nM. FIG. 2 shows the inhibition activity of myristoylated truncatedpeptides. Results obtained with non-myristoylated peptides are displayedin Table 1 (infra).

It appears that peptide Myr 2-48 shows the highest inhibitory activity.The larger peptides, Myr 2-68 and Myr 2-78, although very efficient at800 nM are less active at lower doses. The smaller peptide Myr 2-28 islargely less active although a 50% inhibition is observed at 800 nM.

As some activity still persists for the peptide smaller than 2-48, inorder to evaluate the contribution of the N-terminal amino acids, wehave produced and evaluated a new set of short peptides. The results areshown on FIG. 3. From this figure it is obvious that Myr 2-38 and Myr2-28 peptides still retain a significant inhibitory activity as theyalmost completely blocks HBV infection at 8 μM. By contrast the 2shorter peptides are no longer active and the shorter one tends toincrease HBV infectivity, for unknown reasons.

The effect of myristoylation and C-terminal truncation of pre-S1peptides was also studied through RNA analysis. The results confirm thatmyristoylated truncated peptides displayed enhanced inhibitory activityas compared to the corresponding non myristoylated peptides, and thatthe highest inhibitory activity is obtained with Myr 2-48.

c) HBV Infection Inhibitory Activity of Pre-S1 Homologous Sequences

A recently discovered primate hepadnavirus, the Wolly Monkey Hepatitis Bvirus (WMHBV) has been shown very poorly infectious for Chimpanzee andfor human hepatocyte primary cultures. The WMHBV pre-S1-78 polypeptidicsequence shows 66% sequence identity to the original HBV derivedpeptide. We have investigated the inhibitory activity of a WMHBV derivedMyr 2-48 peptide (SEQ ID NO: 14) towards HBV infection of human cells.

FIG. 4 illustrates the comparative inhibitory activity of WMHBV and HBVderived myristoylated 2-48 peptides (64% sequence identity). Thisexperiment clearly shows that the WMHBV derived peptide is surprisinglynearly as efficient as the HBV derived peptide in inhibiting the HBVinfection. This result is in contrast with the complete absence ofactivity of a DHBV derived peptide (PreS Myr 2-41 (SEQ ID NO: 16), seetable 1) on HBV infection although this peptide is a strong inhibitor ofDHBV infection.

From these results we can conclude that it is possible to efficientlyinhibit the HBV infection. The tolerance of up to 46% variations in thepeptide sequence suggests that it will be possible to inhibit theinfection of HBV viruses of all genotypes with a single peptide.

TABLE 1 summary of the results of the competition experiments.HBV infectivity Non myristoylated of human MyristoylatedHBV infectivity of polypeptides hepatocytes polypeptideshuman hepatocytes (100 μM) inhibition (1 μM) inhibition HBV PreS 1-78 NDHBV Myr PreS 2-78 ++++ (SEQ ID NO: 1) (SEQ ID NO: 4) HBV PreS 1-68 NDHBV Myr PreS 2-68 ++++ (SEQ ID NO: 5) HBV PreS 1-48 +++HBV Myr PreS 2-48 +++++ (SEQ ID NO: 6) (SEQ ID NO: 7) HBV PreS 1-38 NDHBV Myr PreS 2-38 +++ (SEQ ID NO: 8) HBV PreS 1-28 + HBV Myr PreS 2-28++ (SEQ ID NO: 9) (SEQ ID NO: 10) HBV PreS 1-18 ND HBV Myr PreS 2-18 −(SEQ ID NO: 11) HBV PreS 1-8 ND HBV Myr PreS 2-8 − (SEQ ID NO: 12)HBV PreS 19-48 − HBV Myr PreS 19-48 − (SEQ ID NO: 17) (SEQ ID NO: 18)WMHBV PreS 1-48 +++ WMHBV Myr PreS 2-48 +++++ (SEQ ID NO: 13)(SEQ ID NO: 14) DHBV PreS 1-41 − DHBV Myr PreS 2-41 − (SEQ ID NO: 15)(SEQ ID NO: 16) ND: Not Determined

EXAMPLE 3 In Vivo DHBV Infection Inhibition with Pre-S-HBV SyntheticPolypeptides

Ducks are simultaneously injected with DHBV and duck preS Myr 2-41(DpreS2-41^(myr)), heron preS Myr 2-44 (HepreS2-44^(myr)), human preSMyr 2-68 (HupreS2-68^(myr)), duck preS Myr 2-21 (DpreS2-21^(myr)) orddH₂O. The viremia of infected animals is assessed by Dot Blot analysisof viral DNA, 5, 9, 15 and 28 days post-infection. The results displayedin FIG. 5 show that viral DNA is detected in control animals treatedwith ddH₂O, indicated a successful infection of these animals. On thecontrary, a transitory viremia can be observed at days 9 and 15post-infection in ducks treated with either DpreS2-41^(myr) orDpreS2-21^(myr). At day 28, viral DNA is decreased or no longerdetectable which suggests that the animals eliminated the virus. Thisanalysis is further supported by the Western Blot analysis shown in FIG.6 that shows the L protein of DHBV is not detected in the serum of duckstreated with either DpreS2-41^(myr) or DpreS2-21^(myr) 35 dayspost-infection.

On the contrary, the human peptide HupreS2-68^(myr) seems to delay butdo not prevent infection of ducks with DHBV (FIGS. 5 and 6).

Since peptide DpreS2-21^(myr) was shown to have no in vitro inhibitoryactivity towards DHBV infection of primary duck hepatocytes, the in vivoprotection observed with this peptide would result from an indirecteffect of the peptide, i.e. enhancement of the immune response througheliciting antibodies directed against DpreS2-21^(myr).

This experiment illustrates that myristoylated synthetic peptides canconfer protection against hepatitis B virus infection.

The following citations are incorporated herein by reference:

-   Breiner et al. (1998). Carboxypeptidase D (gp180), a Golgi-resident    protein, functions in the attachment and entry of avian hepatitis B    viruses. J. Virol. 72:8098-8104;-   DeMeyer et al. (1997). Organ and species specificity of hepatitis B    virus (HBV) infection: a review of literature with a special    reference to preferential attachment of HBV to human hepatocytes. J.    Viral Hepat. 4, 145-153;-   Gausepohl, H. et al. (1989). Int. J. Prot. Pept. Res. 34, 287-294.-   Gausepohl, H. (1990). Peptidsynthesen an Polystyrol-Polyoxyethylen    Pfropfcopolymeren im Vergleich zu anderen Trägern in der    Festphasensynthese nach dem Durchflussprinzip, PhD thesis University    of Tübingen.-   Gripon, P., C. et al. (1988). Hepatitis B virus infection of adult    human hepatocytes cultured in the presence of dimethyl sulfoxide.    Journal of Virology. 62:4136-43.-   Gripon, P., et al. (1993). Reproducible high level infection of    cultured adult human hepatocytes by hepatitis B virus: effect of    polyethylene glycol on adsorption and penetration. Virology.    192:534-40.-   Guguen-Guillouzo et Guillouzo (1986). Methods for preparation of    adult and fetal hepatocytes. p 1-12. In A. Guillouzo and C.    Guguen-Guillouzo (ed.), Isolated and cultured hepatocytes. Les    Editions INSERM Paris. John Libbey and Co, Ltd., London, United    Kingdom.-   Kuroki et al. (1994). A cell surface protein that binds avian    hepatitis B virus particles. J. Virol. 68:2091-2096;-   Le Seyec et al. (1998). Role of the preS2-domain of the large    envelope protein in hepatitis B virus assembly and infectivity. J.    Virol., 72, 5573-5578.-   Le Seyec et al. (1999). Infection process of the hepatitis B virus    depends on the presence of a defined sequence in the    pre-S1-domain. J. Virol., 73, 2052-2057.-   Nassal et al. (1993). Hepatitis B Virus replication. Trends    Microbiol. 1, 221-228.-   Nassal et al. (1996). H. Hepatitis B virus replication, an    update. J. Viral Hepatitis, 3, 217-226.-   Root, M. J. et al. (2001). Science, 291:884-888.-   Schlicht et al. (1987). Biochemical and immunological    characterization of the duck hepatitis B virus envelope proteins. J.    Virol. 61:2280-2285.-   Urban et al. (1998). Avian hepatitis B virus infection is initiated    by the interaction of a distinct preS-subdomain with the cellular    receptor gp180. J. Virol. 72:8089-8097.-   Urban et al. (2000). Receptor recognition by a hepatitis B virus    reveals a novel mode of high affinity virus-receptor interaction.    EMBO J. 19 (6):1217-1227.-   Urban et al. (1998). Avian hepatitis B virus infection is initiated    by the interaction of a distinct preS-subdomain with the cellular    receptor gp180. J. Virol. 72: 8089-8097.-   Sambrook, J., E. F. Fritsh, and T. Maniatis. (1989). Molecular    cloning, a laboratory manual. Cold Spring Harbor Laboratory Press,    New York

1. A method for inhibiting HBV infection of a cell in a subject,comprising administering to the subject a synthetic polypeptide offormula (I)X-Y-Z  (I) wherein X is an amino acid, or absent; Y is the amino acidsequence consisting of at least amino acids 2 to 28 of pre-S1 region ofHBV large (L) envelope protein corresponding to SEQ ID NO:2; Z, linkedto the —CO— group of the last residue of Y, is the amino acid sequenceconsisting of at least one and at most 30 consecutive amino acids ofpre-S1 region of HBV L envelope protein corresponding to SEQ ID NO:3, orabsent; said polypeptide being chemically modified to bear a hydrophobicmoiety.
 2. The method according to claim 1, wherein the hydrophobicmoiety is a saturated or unsaturated fatty acid having at least 4 carbonatoms.
 3. The method according to claim 1, wherein the hydrophobicmoiety is myristic acid or stearic acid.
 4. The method according toclaim 1, wherein the first amino acid of said polypeptide is chemicallymodified to bear a hydrophobic moiety.
 5. The method according to claim1, wherein said HBV L envelope protein is selected from a groupconsisting of HBV L envelope protein of human HBV, chimpanzee HBV strainLSH, woodchuck HBV, and Woolly Monkey HBV.
 6. The method according toclaim 5, wherein said human HBV is HBV strain alpha1.
 7. The methodaccording to claim 5, wherein said human HBV is HBV subtypes adr, ad,adw, adyw, ar or ayw.
 8. The method according to claim 1, wherein thepolypeptide has the amino acid sequence selected from a group consistingof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13,and SEQ ID NO:
 14. 9. The method according to claim 1, wherein thepolypeptide consists of SEQ ID NO:
 2. 10. The method according to claim9, wherein the first amino acid of the polypeptide of SEQ ID NO: 2 ischemically modified to bear myristic acid or stearic acid.